The Role of Osr1 in VUR Pathogenesis in Mice and Humans

Marie-Lyne Fillion1, Christine L. Watt1, Jasmine El Andalousi2, Fatima Tokhmafshan1, John-Paul Capolicchio3, Mohamed El-Sherbiny3 Roman Jednak3 and Indra R. Gupta1,2

1. Department of Human Genetics, McGill University, Montreal, Quebec, Canada; 2. Department of Pediatrics, McGill University Health Center, Montreal, Quebec, Canada; 3. Division of Urology, Montreal Children’s Hospital and McGill University, Montreal, Quebec, Canada

Background: Vesico-ureteric reflux (VUR), the retrograde flow of urine from the bladder to the kidneys, is a congenital abnormality that predisposes children to urinary tract infections and renal failure. Normally, the bladder and ureter muscle occlude the uretero-vesical junction (UVJ) during voiding and prevent the reflux of urine, but in VUR, this mechanism is defective. Odd Skipped Related 1 (Osr1) is a zinc finger transcription factor that, if knocked out, causes lethal cardiac defects and failure of kidney development. Osr1 is expressed in the undifferentiated metanephric mesenchyme, where it promotes cell survival, and in the mesenchyme of the primordial bladder, where it could play a similar role. Osr1-expressing cells in the intermediate mesoderm have been shown to contribute to the ureteral muscle cell lineage. Thus, Osr1 haploinsufficiency could affect the integrity of the UVJ.

Hypothesis: Osr1 haploinsufficiency may lead to higher levels of apoptosis in the mesenchyme adjacent to the nephric duct and in the developing bladder resulting in failure of ureter elongation and/or smooth muscle development, either of which could result in a refluxing UVJ.

Results: We are using an Osr1+/- mouse model, maintained on the C57Bl/6J (B6) background, whose null allele was created by inserting a LacZ cassette into exon 2. These mice appear to have grossly normal urinary tract development and form kidneys. β-galactosidase activity was assessed as a reporter of Osr1 expression in heterozygous Osr1 mice. At E9.5, β-galactosidase activity was observed in the nephric mesenchyme and became restricted to the undifferentiated cap mesenchyme as kidney development progressed. β-galactosidase activity was also observed in the ureteric and bladder mesenchyme and in the smooth muscle layer of the bladder at the newborn stage. Osr1+/- newborn mice were tested for VUR and exhibited a 20.8% incidence of VUR compared to 2.9% in wildtype littermates. Osr1+/- mice also exhibited reflux at much lower intravesical pressures than the few wildtype mice with VUR, suggesting a more severe UVJ defect. The intravesical ureter length (IVU) was measured by injecting red cresol dye into the renal pelvis. Osr1+/- mice were found to have shorter IVUs than wildtype littermates. Inspection of urinary tract sections stained with hematoxylin and eosin and of Osr1+/-XB6tg(Hoxb7-EGFP) offspring, which express green fluorescent protein (GFP) in the nephric duct and its derivatives, by fluorescent microscopy revealed that Osr1 heterozygotes also exhibit duplex or bifid collecting systems and hydronephrosis. No renal malformations were observed in Osr1 heterozygotes, as assessed by kidney planar surface area, kidney weight and renal histology when compared to wildtype mice. Fluidigm sequencing was performed using DNA from 95 children with VUR to screen for OSR1 gene mutations. Two heterozygous missense variants were identified in two different patients, E258Q and G165E. Both of these variants are in close proximity to zinc finger motifs and are predicted to be deleterious to protein function by Polyphen-2. A L218L synonymous variant was also identified in the 2nd zinc finger of OSR1 and was seen in 5 patients. This SNP has previously been associated with smaller kidney size in newborns and is predicted to reduce spliceosome binding affinity to the second exon of OSR1, resulting in a decrease in mRNA stability and a loss of function.

Conclusion: Heterozygous mutations in Osr1/OSR1 are implicated in the pathogenesis of VUR in mice and men.